Method for inhibiting brain tumor or colon carcinoma

ABSTRACT

A method for inhibiting cell growth is provided. The method includes the step of systemically administering a pharmaceutical preparation containing an interferon as an active ingredient two or more times a day, and is specifically useful for cancer treatment.

TECHNICAL FIELD

The present invention is related to a method for inhibiting cell growthintended for especially tumor cells, which is useful clinically andexperimentally.

BACKGROUND ART

According to the vital statistics of Japan in 1994, the deceased numberby cancer increases 7878 people from the previous year and is 244000people. In this way, the deceased number by cancer has held the top spotsince 1981 and occupies 28% of the total number of deaths. When thesubjects are limited to the 50 from the 40 years old, the deceasednumber by cancer amounted to about one third of the total deceasednumber, and the number of cancer patients was 1327000 in the year of1993. Recently, a variety of anticancer drugs and therapy have beendeveloped, but the cancer is the big subject that still should betackled.

The application the interferon has been tried to various cancers, inworld every country heretofore. At present, the efficacy of interferonis seen in, for example, hairy cell leukemia, Kaposi sarcoma, chronicmyeloid leukemia, non-Hodgkin's lymphoma, multiple myeloma, malignantmelanoma, and renal cell carcinoma (Tyringt et al., “Interferon” pp.399-308, edited by Baron et al.).

The application of the various kinds of interferon such as interferon-α,interferon-β and interferon-γ as the anticancer drug were tried by asmany as 18 research groups, under the guidance of the Ministry of Healthand Welfare “the Special Research regarding the Clinical application ofInterferon” from the latter half of the 1970's even in Japan.

About these results, according to the report of Ohno (“Gan ToKagakuryoho”, vol. 14, No. 5, pp. 1194-1202, 1987), the efficacy to therenal cell cancer is 6-23% with interferon-α and 4-20% withinterferon-β; to the brain tumor in systemic administration is 4-20%with interferon-α and 13-17% with interferon-β; to the multiple myelomais 0-30% with interferon-α and 4-25% with interferon-β; to the skincancer in local administration is 48-72% with interferon-α and 33-53%with interferon-β. The approval of the application of these drugs hasbeen obtained on the basis of these results. Additionally, interferon isalso effective to hematopoietic tumors. Especially, interferon-α iseffective to chronic lymphocytic leukemia in 3 cases of 19 cases; iseffective to chronic myeloid leukemia in 4 cases of 14 cases; and iseffective to hairy cell leukemia in 2 cases of 3 cases.

Unlike these results, the efficacy which is 0-6% to the solid tumor suchas mammary carcinoma, stomach cancer and hepatocellular carcinoma hasbeen hardly admitted.

Since interferon is certainly effective to renal cell cancer and hairycell leukemia, which are not sensitive to the other anticancer drugs andexhibits some effects on multiple myeloma and malignant lymphoma onwhich the other drugs are ineffective, the efficacy of interferon isadmitted. However, the results are still not satisfactory, since theinterferon have almost no efficacy on solid tumors and the efficacythereof is still around 20%, except the local administration of thedrugs to skin cancer.

Efficacy of the interferon as therapeutic drugs for hepatitis B andhepatitis C is also approved based on antiviral actions thereof, as wellas the applications based on antitumor effect thereof. It is known thathepatitis C causes chronic hepatitis and progress to liver cirrhosis,hepatocellular carcinoma in succession. It has been clarified that 80percent of hepatocellular carcinoma occurs based on chronic hepaticdiseases caused by the hepatitis C virus. The interferon has anti-viralactivities to these hepatitis C viruses and allow the patients to befree from the virus and allow transaminase to be normal, and have becomethe first choice of therapy of hepatitis C (Ikegami et al., “Igaku NoAyumi”, vol. 181, No. 5, pp. 341-344, 1997).

However, recent reports show that the effects of interferon are greatlyaffected by the amount of hepatitis C virus, that is, efficacy cannot besignificantly obtained in hepatitis C patients with a large amount ofthe virus. Accordingly, how to improve the efficacy for these patientstherefore becomes a big subject (Yatsuhashi et al., “Igaku No Ayumi”,vol. 181, No. 5, pp. 333-336, 1997).

Okushin et al. indicated the possibility of a new approach to theseproblems (“Kanzo”, vol. 36, pp. 735, 1995: and “Kanzo”, vol. 38, No. 1,1997). Specifically, they found that therapeutic results for hepatitis Cpatients that are not effective for conventional therapies can bemarkedly improved when an interferon-β is administered to such hepatitisC patients twice a day while a daily dose is divided into two doses perday.

The twice-a-day administration increases the effects to the virusreplication, and additionally, increases antitumor activities, asreported by Tanahashi et al. (“Gan To Kagakuryoho”, vol. 14, No. 4, pp.1156-1159, 1987) and Niijima et al. (Cancer Immunology, Immunotherapy,vol. 30, No. 2, pp. 81-85, 1989). Namely, they administered agenetically recombinant human interferon-β to the urinary bladder cavityof patients with superficial bladder tumor at a dose of 3600×10⁴ once aday or at a dose of 1800×10⁴ twice a day, and evaluated the antitumoreffects of the administration. Consequently, a higher efficacy can beobtained in the administration at a dose of 1800×10⁴ twice a day than inthe administration at a dose of 3600×10⁴ once a day.

As described above, the reports on antiviral action or antitumor actionshow that the efficacy becomes higher in the administration of aninterferon twice a day than in the administration in the same amountonce a day. Although the mechanisms of antiviral and anti-tumor actionof interferon have not been clarified yet, it is speculated that theantiviral action and anti-tumor action act under different mechanismsfrom each other, (Gewert et al., “Interferon”, pp. 289-297, edited byBaron et al.; Fleischmann et al., “Interferon”, pp. 299-309, 1992,edited by Baron et al.).

Since the effects on the anti-tumor activity have been obtained at avery high dose of 1800×10⁴ units per dose per day and have been obtainedwith local administration, the effects with systemic administration suchas intravenous administration have not been clarified yet. Inparticular, it is known that such interferon rapidly disappears from theblood circulation when they are intravenously administered (Satoh etal., “J. Interferon Res”, vol. 4, No. 3, pp. 411-422, 1984).

As mentioned above, since the sufficient therapy or drug therapy havenot been established yet in treatment of disease based on the abnormalproliferation of the cell, especially cancer, the establishment of thenovel therapy and also drug therapy are hoped from now on.

Accordingly, an object of the present invention is to provide anindustrially and medically useful novel method for inhibiting cellgrowth, which is directed to the therapy of cancers and of diseasesoccurred under a similar action mechanism, on which the interferon havenot yet exhibited sufficient therapeutic effects.

DISCLOSURE OF INVENTION

The present invention provides a method for inhibiting cell growth,which includes the step of systemically administering a pharmaceuticalpreparation containing an interferon as an active ingredient two or moretimes a day.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing differences in growth inhibitory effect onbrain tumor among different medication methods of an interferon.

FIG. 2 is a graph showing differences in growth inhibitory effect oncolon carcinoma among different medication methods of an interferon.

BEST MODE FOR CARRYING OUT THE INVENTION

Interferon for use in the present invention may be any of interferon-α,-β, and -γ, and interferon of consensus type and hybrid type and may bederived from any of natural interferon, genetically recombinantinterferon and chemically synthesized interferon. A geneticallyrecombinant interferon-β and natural interferon-β are preferably used,of which a natural interferon-β is most preferable.

In the preparation of such interferon using the genetic recombinationtechnologies, host cells include, for example, CHO (Chinese hamsterovary) cells, mouse C127 cells, and other mammalian cells: cells ofinsects such as silk worm and armyworms (Mamestra brassicae); andmicroorganisms such as Escherichia coli, Bacillus subtilis, and yeasts.Additionally, mice, rats, hamsters, rabbits, goats, sheep, pigs andcattle, for example, can also be used.

The interferon thus prepared can be purified and isolated from aconditioned medium if a cell culture, insect extract, microbial extract,and organism extract by a variety of chromatographic techniques. Suchchromatography for use herein is not specifically limited as far as ithas affinity to the interferon and includes, for example, a silicondioxide (silica) or calcium phosphate column, metal chelate column, ionexchange column, and gel filtration column.

Separately, a natural interferon-β is generally prepared, for example,in the following manner. Interferon-β producing cells are cultured onthe surface of, for example, a glass or plastic or on the surface of amicrocarrier made of DEAE-dextran and are then subjected to inductiontreatment with, for example, a synthetic double-strand RNA such as PolyI:C and sequentially to super induction treatment (e.g., metabolicinhibition process by the combination use of cycloheximide andactinomycin D, or ultraviolet irradiation process), and the resultingcells are cultured in a culture medium for 20 to 48 hours, and aninterferon-β is produced and is obtained as a culture medium containinginterferon-β.

The interferon-β in the culture medium thus obtained is generally low inconcentration, and the culture medium contains a variety of contaminantsderived from cells or additives. The interferon-β must be concentratedand purified for medicaluse. A technique for the concentration andpurification of interferon-β is not specifically limited, but ispreferably a chromatographic technique using an insoluble carrier bondedwith a blue dye and a metal-chelate-bonded carrier. Specifically, in thetechnique, a crude interferon-β-containing mixture is brought intocontact with the insoluble carrier bonded with a blue dye, and theinterferon-β is recovered as a solution using an eluant, and theinterferon-β solution is brought into contact with themetal-chelate-bonded carrier in which a metal such as zinc is chelated,and the interferon-β is recovered using an eluant to thereby yield aconcentrated and purified interferon-β.

The interferon for use in the present invention can be administered, asit is or as a pharmaceutical composition containing a carrier andexcipient which are pharmaceutically allowable.

Formulation for administration can be prepared by a known method. Forexample, the interferon is generally dissolved in a sterile aqueoussolution for use in injections, or is suspended in an extract, or isfurther emulsified to be embedded in liposome. A solid preparation canbe prepared by a known method, for example, by a method in which anexcipient such as mannitol, trehalose, sorbitol, lactose, glucose orraffinose is added to the interferon, and the resulting mixture isfreeze-dried to yield a lyphilized preparation. Further, thislyophilized preparation can be powdered and is used as a powder. Theresulting powder can be used as a solid by mixing the powder with, forexample, polylactic acid or glycolic acid. A gel (gelatinizedpreparation) can be prepared by a known method such as a method in whichthe interferon is dissolved in a thickener or polysaccharide. Suchthickeners include, for example, glycerol, polyethylene glycol,methylcellulose, carboxymethylcellulose, hyaluronic acid, andchondroitin sulfate.

Each of these pharmaceutical preparations may further comprise astabilizer such as human serum albumin, human immunoglobulinα2-macroglobulin, and an amino acid; and a dispersing agent orabsorbefacient such as an alcohol, sugar alcohol, ionic surfactant andnonionic surfactant, within a range not deteriorating the bioactivitiesof interferon. Additionally, the pharmaceutical preparations may furthercomprise a trace metal or an organic acid salt, if necessary.

The purified interferon-β preparation thus obtained is formulated intothe aforementioned dosage form and can be used as a therapeutic agentwhich is useful in therapeutic treatment or prophylactic treatment ofdiseases due to abnormal cell growth and for which therapy and drugtherapies have not yet been established, especially in diseases in whichthe target cell is a tumor cell of, for example, brain tumor or coloncarcinoma.

A feature of the invented method is that a pharmaceutical preparationcontaining the interferon as an active ingredient is systemicallyadministered two or more times a day for the inhibition of cellproliferation. Administration may be any of, for example, intravenousadministration, intramuscular administration and subcutaneousadministration, of which intravenous administration is preferablyemployed. As an administration schedule, the pharmaceutical preparationcan be administered any times a day as far as it is administered two ormore times a day, but is preferably administered twice a day. As anadministration interval, the pharmaceutical preparation can beadministered continuously two or more times a day or at intervals ofnearly 24 hours (0 hr<administration interval<24 hr), but is preferablyadministered at intervals of from 1 hr to 23 hr.

A dose can be appropriately determined depending on, for example, theage and body weight of the patient, the condition of the patient,administration form, and administration route, and is generally in arange from 1×10⁴ to 10000×10⁴ units per dose per day, and preferably ina range from 10×10⁴ to 1700×10⁴ units per dose per day.

The present invention will be illustrated in further detail withreference to several examples below, which are not intended to limit thescope of the invention.

EXAMPLES Example 1 and Comparative Example 1

A tumor block of human brain tumor U 251-SP was subcutaneouslytransplanted to nude mice (Balb/c nu/nu). After confirming the growth(proliferation) of the tumor (16 days after transplantation), a placeboand a natural human interferon-β (“Feron” available from TorayIndustries, Inc.) were administered to the mice every day. In a controlgroup, the placebo was administered to the mice via tail vein twice aday in the morning and evening. The interferon-β was administered at adose of 100×10⁴ international units per individual once a day, and at adose of 50×10⁴ international units per individual twice a day in themorning and evening. In the once-a-day administered group at a dose of100×10⁴ international units per individual, the interferon-β wasadministered in the morning and the placebo was administered in theevening.

The size of tumor was determined over time using micrometer calipers,and the volume of tumor (mm³) was calculated according to the followingequation: Tumor volume (mm³)=Major axis (mm)×Minor axis (mm)²/2 (as meanamong eight mice per group).

FIG. 1 is showing the difference in the growth inhibitory effect by thedifference of the administration protocol of interferon. The ordinateshows the tumor volume that was calculated from tumor diameter and theabscissa indicates the number of the days after tumor implantation Thetumor growth in the once-a-day administered group (closed triangle) at adosage of 100×10⁴ international units interferon-β of per head wassignificantly suppressed about 50% to 60% of that in the control group(open circle) at 30 days after tumor implantation. In contrast, thetumor growth in the twice-a-day administered group (closed square) at adosage of 50×10⁴ international units of interferon-β per head was moresignificantly suppressed about 15% to 25% compared to that in theonce-a-day administered group at a dosage of 100×10⁴ international unitsinterferon-β per head, even through the total doses per day were equalin the two groups, and about 10% to 15% compared to that in the controlgroup. (*: p<0.05, **: P<0.01, ***: p<0.001, versus the control group).

Example 2 and Comparative Example 2

A tumor block of human colon carcinoma LS 180 was implanted to nude micesubcutaneously (Balb/c nu/nu). After confirming the growth(proliferation) of the tumor (15 days after inoculation), placebo andnatural human interferon-β (“Feron” available from Toray Industries,Inc.) were administered to the mice every day. In a control group, theplacebo was administered to the mice via tail vein twice a day in themorning and evening. The interferon-β was administered at a dosage of100×10⁴ international units per head once a day, and at a dosage of50×10⁴ international units per individual twice a day in the morning andevening. In the once-a-day administered group at a dosage of 100×10⁴international units per head, the interferon-β was administered in themorning and the placebo was administered in the evening.

The size of tumor was determined sequentially using micrometer calipers,and the volume of tumor (mm³) was calculated according to the followingequation: Tumor volume (mm³)=[Major axis (mm)]×[Minor axis (mm)]²/ 2 (asmean among five mice per group).

FIG. 2 is showing the difference of the growth inhibitory effect by thedifference of the administration protocol of interferon with theproliferation rate at 30 days after tumor inoculation. The tumor volumein the once-a-day administered group (hatched column) at a dosage of100×10⁴ international units interferon-β per mouse was suppressed about10% compared to that in the control group (open column). In contrast,the tumor volume in the twice-a-day administered group (closed column)at a dosage of 50×10⁴ international units of interferon-β per mouse wassuppressed about 15% compared to that in the once-a-day administeredgroup at a dosage of 100×10⁴ international units interferon-β per mouse,even through the total dosages per day were equal in the two groups, andabout 25% compared to that in the control group.

INDUSTRIAL APPLICABILITY

The invented method can improve the anti-tumor effects remarkably andcan provide effective means to cancer treatment.

What is claimed is:
 1. A method for inhibiting brain tumor or coloncarcinoma cell growth comprising systemically administering apharmaceutical preparation including an interferon-β as an activeingredient two or more times a day.
 2. A method for inhibiting braintumor or colon carcinoma cell growth according to claim 1, wherein saidinterferon is a natural interferon.
 3. A method for inhibiting braintumor or colon carcinoma cell growth according to claim 1, wherein theinterferon is administered at a dosage range from 10×10⁴ to 1700×10⁴units per administration.